Low substituted polymyxins and compositions thereof

ABSTRACT

The present invention relates to novel low substituted polymyxins and compositions thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of PCT/EP2015/064764,filed on Jun. 29, 2015, which claims the benefit of U.S. ProvisionalApplication No. 62/022,399, filed on Jul. 9, 2014, both of which areincorporated by reference in their entirety herein.

The present invention relates to novel low substituted polymyxins andcompositions thereof.

BACKGROUND

Polymyxins were discovered in 1947 as antibiotics produced by Bacilluspolymyxa. Polymyxins are antibiotic decapeptides containing aheptapeptide ring and a N-terminal amide coupled fatty acid. Today, twocommercial Polymyxin mixtures are in clinical use; Polymyxin B andPolymyxin E (Colistin). Both mixtures comprise a variety of componentsas described in J Chromatogr A. 2002 Nov. 8; 976(1-2):65-78 by Goevaertset al and in Talanta 2011 Feb. 15; 83(5):1521-9 by Van den Bossche etal. According to the European pharmacopoeia, Colistin should comprisemore than 77% of Polymyxin E1, E2, E3, E1-i and E1-7MOA, but less than10% of each of the minor components Polymyxin E3, E1-i and E1-7MOA.

Due to toxicity associated with Colistin, the mixture was improved bysulfomethylation in the 1950'ties. The sulfomethylated Colistin iscalled Colistimethate sodium (CMS) which has been considered to be aprodrug of Colistin. CMS is still in clinical use as a last-linetreatment option for multidrug-resistant organisms such as Pseudomonasaeruginosa, Acinetobacter baumannii, Klebsiella pneumonia and other Gramnegative pathogens. For many years, solutions of CMS have also beenadministered by nebulization into the lungs of patients with cysticfibrosis (CF) to manage colonization or infections caused by P.aeruginosa.

Today, the increasing problem with infections caused by antibioticresistant pathogens has resulted in an increased use of products such asColistin.

The fact that commercial CMS products contain a complex mixture ofderivatives of different Polymyxins has several unfortunateconsequences. The foremost of these relates to the therapeutic value ofany marketed product. Since CMS may be considered a antibiotic-reservoironce injected or inhaled into the body, it is of importance thattherapeutic levels of the activated compounds are reached before renalor metabolic clearance. Thus, a sulfomethylated polymyxin containing twosulfomethyl substituents only, provides a well-defined product withrespect to molecular weight and charge.

An object of the present invention is therefore to provide improvedpolymyxin compounds and compositions useful as antimicrobial agents, andto overcome or at least mitigate the disadvantages of the prior artproducts.

SUMMARY OF THE INVENTION

This invention concerns polymyxins which comprise a total of twosulfomethyl groups attached to the γ-amino groups on DAB or DAP residuesin the polypeptide moiety of polymyxins. These compounds andcompositions thereof can be used in treatment of bacterial infections.

In one aspect, the present invention provides new polymyxins which aremore water soluble than Polymyxin E and Polymyxin B. In yet anotheraspect, the present invention provides sulfomethylated polymyxins whichare more stable in aqueous solution than the marketed CMS. In yetanother aspect, the present invention provides mixtures ofsulfomethylated polymyxins which are more uniform than the marketed CMS.Finally, the present compounds, or the mixtures or compositions thereof,provides for an improved polymyxin dosing and therapy compared with theprior art polymyxin compositions, which is currently not well-defined.

Thus, the present invention provides a novel polymyxin represented byformula (I):

wherein

-   R¹ is an aliphatic linear or branched C₆-C₁₀ acyl group, or

-   R⁵ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, or —CH₂C₆H₅;-   R⁶ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, or —CH(CH₃)CH₂CH₃;-   each of R², R³, R⁴, R⁷ and R⁸ is either —(CH₂)_(x)CH₂NH₂ or    —(CH₂)_(x)CH₂N(CH₂SO₃M)₂;-   wherein x is 0 or 1;-   wherein M is a cation; and-   provided that one of R², R³, R⁴, R⁷ and R⁸ are    —(CH₂)_(x)CH₂N(CH₂SO₃M)₂.

According to one embodiment of the present invention, polymyxins areprovided, represented by the formula (I); wherein two sulfomethyl groupsare attached to the same γ-amino group.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is —CH₂CH(CH₃)₂;-   each of R³, R⁴, R⁷ and R⁸ is —CH₂CH₂NH₂;-   and;-   R² is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is —CH₂CH(CH₃)₂;-   each of R², R⁴, R⁷ and R⁸ is —CH₂CH₂NH₂;-   and;-   R³ is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is —CH₂CH(CH₃)₂;-   each of R², R³, R⁷ and R⁸ is —CH₂CH₂NH₂;-   and;-   R⁴ is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is —CH₂CH(CH₃)₂;-   each of R², R³, R⁴, and R⁸ is —CH₂CH₂NH₂;-   and;-   R⁷ is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is —CH₂CH(CH₃)₂;-   each of R², R³, R⁴, and R⁷ is —CH₂CH₂NH₂;-   and;-   R⁸ is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is either —CH₂CH(CH₃)₂ or CH₂C₆H₅;-   each of R³, R⁴, R⁷ and R⁸ is —CH₂CH₂NH₂;-   and;-   R² is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is either —CH₂CH(CH₃)₂ or CH₂C₆H₅;-   each of R², R⁴, R⁷ and R⁸ is —CH₂CH₂NH₂;-   and;-   R³ is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is either —CH₂CH(CH₃)₂ or CH₂C₆H₅;-   each of R², R³, R⁷ and R⁸ is —CH₂CH₂NH₂;-   and;-   R⁴ is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is either —CH₂CH(CH₃)₂ or CH₂C₆H₅;-   each of R², R³, R⁴, and R⁸ is —CH₂CH₂NH₂;-   and;-   R⁷ is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to yet another embodiment of the present invention, polymyxinsrepresented by formula (I) are provided, wherein R¹ is an aliphaticlinear or branched C₆-C₁₀ acyl group;

-   R⁵ and R⁶ is either —CH₂CH(CH₃)₂ or CH₂C₆H₅;-   each of R², R³, R⁴, and R⁷ is —CH₂CH₂NH₂;-   and;-   R⁸ is —CH₂CH₂N(CH₂SO₃M)₂.-   wherein M is a monovalent cation.

According to another aspect of the above embodiments of the presentinvention, R¹ is 6-methyloctanoyl.

According to another aspect of the above embodiments of the presentinvention, R¹ is 6-methyl-heptanoyl.

According to yet another embodiment of the present invention, polymyxinsof formula (I) are provided wherein R¹ is an aliphatic linear orbranched C₆-C₁₀ acyl group, or

-   R⁵ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, or —CH₂C₆H₅;-   R⁶ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, or —CH(CH₃)CH₂CH₃;-   each of R², R³, R⁴, R⁷ and R⁸ is either CH₂NH₂ or CH₂N(CH₂SO₃M)₂;-   wherein M is a cation; and-   provided that one of R², R³, R⁴, R⁷ and R⁸ are CH₂N(CH₂SO₃M)₂.

The present invention furthermore provides a pharmaceutical compositioncomprising one or more of the polymyxins according to the presentinvention.

The composition according to the present invention may furthermorecomprise one or more pharmaceutically acceptable excipients.

According to one embodiment, the pharmaceutical composition according tothe present invention comprises more than 50% as measured by HPLC of thepolymyxins according to the present invention.

According to yet another embodiment, the pharmaceutical compositionaccording to the present invention comprises more than 50% as measuredby HPLC of the polymyxins represented by formula (I).

According to yet another embodiment, the pharmaceutical compositionaccording to the present invention comprises more than 80% as measuredby HPLC of the polymyxins represented by formula (I).

According to yet another embodiment, the pharmaceutical compositionaccording to the present invention comprises more than 90% as measuredby HPLC of the polymyxins represented by formula (I).

According to yet another embodiment, the present composition comprisesmore than 50% as measured by HPLC of one or more of the polymyxinsrepresented by the formulas of FIGS. 1-5.

According to yet another embodiment, the present composition comprisesmore than 80% as measured by HPLC of one or more of the polymyxinsrepresented by the formulas of FIG. 1-5.

According to yet another embodiment, the present composition comprisesmore than 90% as measured by HPLC of one or more of the polymyxinsrepresented by the formulas of FIG. 1-5.

A first embodiment is directed to a polymyxin represented by formula(I):

wherein

-   R¹ is an aliphatic linear or branched C₆-C₁₀ acyl group, or-   R⁵ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, or —CH₂C₆H₅;-   R⁶ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃ or —CH₂CH₂CH₃;-   each of R², R³, R⁴, R⁷ and R⁸ is either —(CH₂)_(x)CH₂NH₂ or    —(CH₂)_(x)CH₂N(CH₂SO₃M)₂;-   wherein x is 0 or 1;-   wherein M is a cation; and-   wherein one of R², R³, R⁴, R⁷ and R⁸ is —(CH₂)_(x)CH₂N(CH₂SO₃M)₂.

In a first aspect of the first embodiment, R1 is heptanoyl,methylheptanoyl, octanoyl, methyloctanoyl, nonanoyl, methylnonanoyl ordecanoyl.

In a second aspect of the first embodiment, R1 is heptanoyl,6-methylheptanoyl, octanoyl, (S)-6-methyloctanoyl, 7-methyloctanoyl,3-hydroxy-6-methyloctanoyl, nonanoyl, (8-methylnonanoyl or decanoyl.

In a third aspect of the first embodiment, the previously describedpolymyxins are characterized in that M is selected from the groupconsisting of Na⁺, K⁺, ½Mg²⁺, H_(m)N(C₁₋₄alkyl)_(n) ⁺, or combinationsthereof, where m is 0-4 and n is 0-4 with the proviso that m+n=4.

In a fourth aspect of the first embodiment, x is 1 and M is H⁺, Na⁺ orK⁺.

In a fifth aspect of the first embodiment, x is 1 and M is Na⁺ or K⁺.

A second embodiment is directed to a composition comprising at least onepolymyxin represented by formula (I):

wherein

-   R¹ is an aliphatic linear or branched C₆-C₁₀ acyl group, or-   R⁵ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, or —CH₂C₆H₅;-   R⁶ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃ or —CH₂CH₂CH₃;-   each of R², R³, R⁴, R⁷ and R⁸ is either —(CH₂)_(x)CH₂NH₂ or    —(CH₂)_(x)CH₂N(CH₂SO₃M)₂;-   wherein x is 0 or 1;-   wherein M is a cation; and-   wherein one of R², R³, R⁴, R⁷ and R⁸ is —(CH₂)_(x)CH₂N(CH₂SO₃M)₂.

In a first aspect of the second embodiment, R1 is heptanoyl,methylheptanoyl, octanoyl, methyloctanoyl, 3-hydroxy-6-methyloctanoyl,nonanoyl, methylnonanoyl or decanoyl.

In a second aspect of the second embodiment, R1 is heptanoyl,6-methylheptanoyl, octanoyl, (S)-6-methyloctanoyl,3-hydroxy-6-methyloctanoyl, 7-methyloctanoyl, nonanoyl, 8-methylnonanoylor decanoyl.

In a third aspect of the second embodiment, the previously describedpolymyxins are characterized in that M is selected from the groupconsisting of Na⁺, K⁺, H_(m)N(C₁₋₄alkyl)_(n) ⁺, ½Mg²⁺ or combinationsthereof, where m is 0-4 and n is 0-4 with the proviso that m+n=4.

In a fourth aspect of the second embodiment, x is 1 and M is H⁺, Na⁺ orK⁺.

In a fifth aspect of the second embodiment, x is 1 and M is Na⁺ or K⁺.

In a sixth aspect of the second embodiment, the pharmaceuticalcomposition comprises more than 50% as measured by HPLC of thepolymyxins represented by formula (I).

In a seventh aspect of the second embodiment, the pharmaceuticalcomposition comprises more than 50% as measured by HPLC of thepolymyxins represented by formula (I) and x is 1 and M is Na⁺ or K⁺.

A third embodiment is directed to a polymyxin represented by formula(I):

wherein

-   R¹ is an aliphatic linear or branched C₆-C₁₀ acyl group;-   R⁵ is —CH₂CH(CH₃)₂ or —CH₂C₆H₅;-   R⁶ is —CH₂CH(CH₃)₂;-   each of R², R³, R⁴, R⁷ and R⁸ is either —CH₂CH₂NH₂ or    —CH₂CH₂N(CH₂SO₃M)₂;-   wherein M is a cation; and-   wherein only one of R², R³, R⁴, R⁷ and R⁸ is —CH₂CH₂N(CH₂SO₃M)₂.

In a first aspect of the third embodiment, the aliphatic C₆-C₁₀ acylgroup or the branched C₆-C₁₀ acyl group optionally contains a hydroxylgroup.

A fourth embodiment is directed to a composition comprising at least onepolymyxin represented by formula (I):

wherein

-   R¹ is an aliphatic linear or branched C₆-C₁₀ acyl group;-   R⁵ is —CH₂CH(CH₃)₂, or —CH₂C₆H₅;-   R⁶ is —CH₂CH(CH₃)₂;-   each of R², R³, R⁴, R⁷ and R⁸ is either —CH₂CH₂NH₂ or    —CH₂CH₂N(CH₂SO₃M)₂;-   wherein M is a cation; and-   wherein only one of R², R³, R⁴, R⁷ and R⁸ is —CH₂CH₂N(CH₂SO₃M)₂.

In a first aspect of the fourth embodiment, the pharmaceuticalcomposition comprises more than 50% as measured by HPLC of thepolymyxins represented by formula (I).

In a second aspect of the fourth embodiment, the pharmaceuticalcomposition comprises more than 80% as measured by HPLC of thepolymyxins represented by formula (I).

In a third aspect of the fourth embodiment, the pharmaceuticalcomposition comprises more than 50% as measured by HPLC of thepolymyxins represented by formula (I) and is used for treatment ofinfections caused by Gram-negative bacteria.

In a fourth aspect of the fourth embodiment, the pharmaceuticalcomposition comprises more than 50% as measured by HPLC of thepolymyxins represented by formula (I) and M is selected frompharmaceutically acceptable cations.

In a fifth aspect of the fourth embodiment, the pharmaceuticalcomposition comprises more than 50% as measured by HPLC of thepolymyxins represented by formula (I) and M is selected frompharmaceutically acceptable monovalent cations.

In a sixth aspect of the fourth embodiment, the aliphatic C₆-C₁₀ acylgroup or the branched C₆-C₁₀ acyl group optionally contains a hydroxylgroup.

A fifth embodiment is directed to a composition comprising at least onepolymyxin represented by formula (I):

wherein

-   R¹ is 6-methyloctanoyl, 6-methylheptanoyl or octanoyl;-   R⁵ is —CH₂CH(CH₃)₂, or —CH₂C₆H₅;-   R⁶ is —CH₂CH(CH₃)₂;-   each of R², R³, R⁴, R⁷ and R⁸ is either —CH₂CH₂NH₂ or    —CH₂CH₂N(CH₂SO₃Na)₂; and-   wherein only one of R², R³, R⁴, R⁷ and R⁸ is —CH₂CH₂N(CH₂SO₃Na)₂.

In a first aspect of the fifth embodiment, the pharmaceuticalcomposition comprises more than 50% as measured by HPLC of thepolymyxins represented by formula (I).

In a second aspect of the fifth embodiment, the pharmaceuticalcomposition comprises more than 80% as measured by HPLC of thepolymyxins represented by formula (I).

In a third aspect of the fifth embodiment, the pharmaceuticalcomposition comprises more than 50% as measured by HPLC of thepolymyxins represented by formula (I) and is used for treatment ofinfections caused by Gram-negative bacteria.

DETAILED DESCRIPTION Brief Description of the Figures

FIG. 1 shows the structure of the disodium salt of PE1-(SM)₂ ¹

FIG. 2 shows the structure of disodium salt of PE1-(SM)₂ ³

FIG. 3 shows the structure of disodium salt of PE1-(SM)₂ ⁵

FIG. 4 shows the structure of disodium salt of PE1-(SM)₂ ⁸

FIG. 5 shows the structure of disodium salt of PE1-(SM)₂ ⁹

FIG. 6 shows a chromatogram of PE1-(SM)₁₀ ^(1,3,5,8,9)

FIG. 7 shows the degradation of PE1-(SM)₁₀ ^(1,3,5,8,9) undernon-equilibrium conditions after 90 min, pH 9.

FIG. 8 shows the degradation of PE1-(SM)₁₀ ^(1,3,5,8,9) undernon-equilibrium conditions after 3 days, pH 8.0.

FIG. 9 shows the degradation of PE1-(SM)₁₀ ^(1,3,5,8,9) undernon-equilibrium conditions after 3 days, pH 7.0.

FIG. 10 shows the degradation of PE1-(SM)₁₀ ^(1,3,5,8,9) undernon-equilibrium conditions after 3 days, pH 6.5.

FIG. 11 shows the degradation of PE1-(SM)₁₀ ^(1,3,5,8,9) undernon-equilibrium conditions after 3 days, pH 6.0.

FIG. 12 shows MS spectra obtained for two substituted Polymyxin E1. m/z677.3 [−2] and 1355.5 [−1] was obtained in negative mode.

FIG. 13 (top) shows a chromatogram confirming the presence of fiveindividual Polymyxin components comprising two sulfomethyl groups (UVsignal at 215 nm).

FIG. 13 (bottom) shows the extracted ion current (EIC) for massescorresponding to mono(bis-sulfomethylated) Polymyxin E1 (m/z of 677.62,1356.2, 1378.06).

FIG. 14 shows mass spectra of mono(bis-sulfomethylated) polymyxin E1.

DEFINITIONS

As used throughout this specification and the appended claims, thefollowing terms have the following meanings:

The term “polymyxin” as described herein refers to antibiotic compoundscomprising a peptide moiety amide-coupled to a fatty acid moiety.

The term “Dab residue” as described herein, refers to a2,4-diaminobutyrate compound amide-coupled to at least one amino acid.The naturally occurring polymyxins usually comprise 6 Dab residues ofwhich 5 have a free γ-amino group.

The term “Dab” as described herein, refers the radical derived from2,4-diaminobutanoic acid, in which the carbon atom adjacent to thecarbonyl carbon (i.e., the α-carbon) has a stereochemistry designated asthe L-configuration. L-Dab is alternatively referred to in theliterature as L-Dbu.

The amino acid in position 6 in the polymyxins represented by formula(I) have D-configuration, i.e. the amino acid residue to which R⁵ isattached.

The term “DAP residue” as described herein refers to a2,3-diaminopropionate compound amide-coupled to at least one amino acid.

The term “DAP” as described herein refers to the compound2,3-diaminopropionate.

The term “sulfomethyl” as described herein refers to the —CH₂SO₃Mmoiety, where M is as defined below. The sulfonate (—SO₃) moiety can bein acidic form, but in a physiologic environment (in vivo) it will havea negative charge and will have an associated cation, such as M.

The term “CMS” as described herein refers to a composition comprisingsulfomethylated Colistin. Chemical abstracts have assigned such acomposition the number 8068-28-8 for CMS.

The term “CMS E1” as described herein refers to a composition made fromsulfomethylation of relatively pure Polymyxin E1 (>80% pure by HPLC).

The term “Colistin” as described herein refers to a compositioncomprising polymyxin E1 and polymyxin E2. Chemical abstracts haveassigned the number 1066-17-7 for Colistin. According to the Europeanpharmacopoeia, Colistin should comprise more than 77% of Polymyxin E1,E2, E3, E1i and E1-7MOA, but less than 10% of each of the minorcomponents Polymyxin E3, E1-i and E1-7MOA.

The term “Polymyxin E” as described herein is used interchangeably with“Colistin”.

The term “Polymyxin E1” as described herein refers to the compoundhaving the CAS no 7722-44-3. Polymyxin E1 is used interchangeably withColistin A.

The term “Polymyxin E2” as described herein refers to the compoundhaving the CAS no 7239-48-7. Polymyxin E2 is used interchangeably withColistin B.

The expression “sulfomethylated polymyxin” as described herein refers toa polymyxin comprising at least one sulfomethyl group attached to aγ-amino group on a L-DAB residue.

The term “M” is a cation and refers to a cationic species containing oneor two positive charges, examples of which include, but are not limitedto Li⁺, Na⁺, K⁺, ½Mg²⁺, H_(m)N(C₁₋₄alkyl)_(n) ⁺, where m is 0-4 and n is0-4 with the proviso that m+n=4.

The expression “an aliphatic linear or branched C6-C10 acyl group,” asdescribed herein, refers to a substituent containing a carbonyl moietyand a non-carbonyl moiety containing a total of 6 to 10 carbon atoms. Itincludes the acyl groups found in known polymyxin compounds. It alsoincludes, but is not limited to, heptanoyl, methylheptanoyl (including(S)-6-methylheptanoyl), octanoyl, methyloctanoyl (including(S)-6-methyloctanoyl, 7-methyloctanoyl), nonanoyl, methylnonanoyl anddecanoyl.

The term “salts” or “salt thereof as described herein, refers to acompound comprising a cation and an anion, which can prepared by anyprocess known to one of ordinary skill, e.g., by the protonation of aproton-accepting moiety and/or deprotonation of a proton-donatingmoiety. Alternatively, the salt can be prepared by either a cation/anionmetathesis or a cation/anion exchange reaction.

The term “more than X % as measured by HPLC” as described herein is tobe understood as the relative integrated area of the pertaining peak(s)in the chromatogram resulting from an HPLC method as described in theexamples of this application or alternatively as described inWO2014195405A1.

According to the present invention, novel polymyxins are providedcomprising two sulfomethyl groups which are attached to any of the Dabresidues of the above structure having a free γ-amine, i.e. any of theDab residues Dab 1, Dab3, Dab5, Dab8 or Dab9. Thus, formula (I) is meantto cover mono(bis-sulfomethylated) polymyxins, but not the tri-, tetra-or penta(bis-sulfomethylated) polymyxins.

An example of the new polymyxins is designated herein as mono(N^(γ)-bis-sulfomethyl) Dab⁹ polymyxin E1, i.e. wherein the substituentsof formula I is represented by R¹ being 6-methyloctanoyl, R⁵ and R⁶ is—CH₂CH(CH₃)₂; each of R², R³, R⁴, and R⁷ is —CH₂CH₂NH₂; and R⁸ is—CH₂CH₂N(CH₂SO₃M)₂ wherein M is Na⁺. An example of such a compound isshown in FIG. 5. The abbreviated name for this compound is PE1-(SM)₂ ⁹.

Other examples of mono bis-sulfomethylated polymyxins are mono(N^(γ)-bis-sulfomethyl) Dab¹ polymyxin E1, mono (N^(γ)-bis-sulfomethyl)Dab³ polymyxin E1, mono (N^(γ)-bis-sulfomethyl) Dab⁵ polymyxin E1, mono(N^(γ)-bis-sulfomethyl) Dab⁸ polymyxin E1, of which the disodiumstructures are shown in FIG. 1-4, respectively.

The expression “—CH₂CH₂NH₂” is understood to cover either —CH₂CH₂NH₂ or—CH₂CH₂NH₃ ⁺ depending on the pH of the medium.

The expression “—CH₂NH₂” is understood to cover either —CH₂NH₂ or—CH₂NH₃ ⁺ depending on the pH of the medium.

The polymyxins as described herein embrace many molecular species. Forexample in aqueous solutions, the charge will depend on pH. Thepolymyxin compounds as described herein cover all pharmaceuticalacceptable salts and ions thereof. Among such polymyxins are of coursethe di-sodium salts. Other pharmaceutically acceptable salts are alsoincluded e.g. potassium, lithium, and ammonium salts (such asH_(m)N(C₁₋₄alkyl)_(n) ⁺ where m is 0-4 and n is 0-4 with the provisothat m+n=4), or combinations thereof.

The polymyxins as described herein may be used in the treatment orprevention of infectious disorders, in particularly infections caused byGram-negative bacteria, such as, but not limited to, Pseudomonasaeruginosa, Klemsiella pneumonia, and Acinetobacter baumannii,Eschericia coli, and Enterobacter aerogenes. According to one aspect,the present invention includes the use of a pharmaceutical compositioncomprising an effective amount of any of the compositions describedherein in the treatment of a gram negative bacterial infection.

The present compounds may be administered in a therapeutically effectiveamount. “A therapeutically effective amount” is meant to describe asufficient quantity of the compound to treat the infection. The skilledperson, will acknowledge that the appropriate daily dosage of aparticular patients may vary depending upon factors such as the disorderbeing treated, and the severity thereof; the composition employed, theage, body weight, sex and diet of the patient, and the preferences andexperience of the medical practitioner involved, etc.

The compositions according to the present invention may be administeredfor use in human as well as veterinary medicine. The compositiondescribed herein may be administered to a patient in need thereof viavarious routes, such as subdermal, by inhalation, oral, parenteral ortopical route. The composition may be in any form well known to theskilled person, and includes e.g. tablets, capsules, powders, granules,lozenges, creams or liquid preparation, such as sterile paerenteralsolutions or suspensions.

In one aspect, the composition is useful for administration byintravenous infusion or injections. An intravenous or injectionformulation may be prepared according to methods well known to theskilled person. For example, the compounds described herein can bedissolved in water for injection, followed by filter sterilization, andfurther followed by transferring said filtered composition to a suitablevial.

According to one aspect, the composition is lyophilized. The lyophilizedpowder is reconstituted, e.g. in water for injection, prior to use, e.g.prior to administration by injection, or prior to administration byinhalation (e.g. using a nebulizer or any other suitable inhalationsdevices). Thus, according to another aspect, the composition isformulated for administration is by inhalation.

The compounds described herein may further comprise one or morepharmaceutically acceptable excipients, including but not limited to apreservative, a buffer, and antioxidant, or a diluent. Suitable diluentsincludes e.g. water for injection, 0.9% NaCl, 5% dextrose in 0.9% NaCl,5% dextrose in water, 5% dextrose in 0.45% NaCl, 5% dextrose in 0.225%NaCl, and Lactated Ringer's solution.

The compounds disclosed herein may furthermore be administered incombination with one or more additional anti-bacterial agents. Thecompounds described herein may when used in combination with anotheranti-bacterial agent be administered simultaneously, separately orsequentially with the one or more other anti-bacterial agent.

The present invention is further illustrated below by way of examples.It is to be understood that the scope of the present invention is in anyway limited by the scope of the following examples.

EXAMPLES

The present disclosure is further supported and exemplified by thefollowing experimental section, but is not to be limited thereto.

Example 1

Starting Material

Isolated polymyxin E1 sulfate (3.5 g) and a 45% w/w aqueous solution ofsodium hydroxymethanesulfonate (11.3 g) were mixed and warmed to 60° C.while stirring. pH was then kept at 7.0-7.5 by several additions of 2 MNaOH. After 18 hr the mixture was cooled to ambient temperature and thecrude product furnished as a white solid by precipitation in 200 mL ofmethanol/acetonitrile 1/1 v/v.

The product was de-salted and polished by the following procedure: A C18column 6μ e.g. Phenomenex X Bridge Prep Shield 10×250 mm or similar waswashed and equilibrated with 5% MeCN (no salt). The column was mountedin a Waters Delta Prep HPLC system, 150 mL/min maximum flow. Thedetector was 5 a Waters 2487 adjusted to 280 nm. A mixture of 9 mL 5%MeCN solution of 230 mg PE1-(SM)₁₀ ^(1,3,5,8,9) and 1 mL 2M NaCl wasloaded the column and the flow was 6-8 mL/min.

The column was eluted and desalted with 5% MeCN with 6-8 mL/min and thePE1-(SM)₁₀ ^(1,3,5,8,9) fraction collected. Some degradation occurredon-column during the process, but by cutting the head and tail off, thehigh purity is maintained. The Head Fraction, ca. 35 mL, was collecteddirectly into 450 mL 100% MeCN and further 550 mL 100% MeCN was addedbefore vacuum-evaporation of the 97:3 MeCN:H₂O solution in a 2 L pearshaped evaporation flask.

The distillation process was performed with a Büchi 15 Rotavapor. The94:6 azeotrope distils off making a water free PE1-(SM)₁₀^(1,3,5,8,9)-residue in the 2 L vacuum-distillation flask. The residuewas removed with 3×8 mL 100% MeOH (dry) and poured into a 50 mL vacuumevaporation flask and vacuum-evaporated to a 1-2 mL MeOH-PE1-(SM)₁₀^(1,3,5,8,9) suspension. 15 mL 100% MeCN was added and the 20solution/suspension was vacuum-evaporated further to dryness with waterbath temperature of 35° C. The pressure was decreased from 70-60 Torrdown to 20-15 Torr during the process. Further vacuum-drying was donefor 30 min with slow rotation in the 35° C. water bath with maintainedvacuum. The yield was 150 mg substance of the PE1-(SM)₁₀ ^(1,3,5,8,9).

Example 2

Production of a Mixture Comprising Mono (N^(γ)-bis-sulfomethyl)Polymyxin E1.

When sulfomethylated polymyxins degrade towards Colistin,hydroxymethane-sulfonate is liberated. The process is reversible as thehydroxymethanesulfonate easily regenerates the parent sulfomethylatedmolecule.

The purpose of this study was to investigate degradation ofsulfomethylated polymyxins under non-equilibrium conditions by thecontinuous removal of hydroxymethanesulfonate using nanofiltration.

The experiment was set up with 2 L 10 mM PE1-(SM)₁₀ ^(1,3,5,8,9) in ROwater and the volume of the solution was kept constant in the filtrationunit by the frequent addition of RO water. The nanofiltration unit wasrun in daily campaigns at 40 C under 3 bar pressure, removing approx. 5l of effluent each time. pH was adjusted with 0.1 M HCl until stabile to8.0, 7.0, 6.5 and 6.0. The results are shown in FIG. 6-11.

MS data confirms di-sulfomethylation of the polymyxins. The FIG. 12shows the MS spectra obtained, m/z 677.3 [−2] and 1355.5 [−1], innegative mode.

Example 3: Synthesis of Low Substituted Colistins

Colistin sulfate (1.0 g, 0.71 mmol) was dissolved in 20 ml water. 45%sodium hydroxymethanesulfonate (0.847 g, 2.841 mmol) and NaHCO₃ (0.12 g,1.40 mmol) was added. The clear solution was stirred for 12 h at 25° C.The product was precipitated by slow addition of 100 ml acetonitrile andmethanol (50:50). The product was isolated as a white powder.

Example 4a: Synthesis of Low Substituted Polymyxin E1

Polymyxin E1 (1.0 g, 0.71 mmol) was dissolved in 20 ml water. 45% sodiumhydroxymethanesulfonate (0.847 g, 2.841 mmol) and NaHCO₃ (0.12 g, 1.40mmol) was added. The clear solution was stirred for 12 h at 25° C. Theproduct was precipitated by slow addition of 100 ml acetonitrile andmethanol (50:50). The product was isolated as a white powder.

Example 4b: Chromatography and MS of Low Substituted Polymyxin E1

Mono(bis-sulfomethylated) Polymyxin E1 as made by example 4a wasanalysed using a Mass Spectrometric (MS in negative mode) and UVcompatible method where separation was performed at pH 6.6. The top ofFIG. 13 shows a chromatogram confirming the presence of five individualPolymyxin components comprising two sulfomethyl groups (UV signal at 215nm). The bottom of FIG. 13 shows the extracted ion current (EIC) formasses corresponding to mono(bis-sulfomethylated) Polymyxin E1 (m/z of677.62, 1356.2, 1378.06).

FIG. 14 shows the individual mass spectra of mono(bis-sulfomethylated)polymyxin E1. The m/z of 677 corresponds to [M+2H]2+, 1356 correspondsto [M+H]+, and 1378 corresponds to [M+Na]+ of mono(bis-sulfomethylated)Polymyxin E1.

Example 5: MIC

A sample comprising ca. 93% mono(N^(γ)-bis-sulfomethyl) Polymyxin E1 wassubjected to MIC analysis against Bordetella bronchiseptica, ATCC 4617and the microbial potency was measured to be 611 μg/g.

MIC Assay

Test organisms were cultured in shakeflasks for approx. 16 hours at 250rpm, 34° C. for Pseudomonas aeruginosa and 37° C. for Klebsiellabaumannii and Acinetobacter baumannii. They were re-cultured to latelog, diluted to concentrations ready to use and frozen with glycerol at÷75° C. The medium used was Mueller Hinton Broth.

Samples were prepared by dissolving 5 mg of the compounds in 1 mlMueller Hinton Broth. The samples were diluted and added to the wells,test organisms were added and after approx. 16 hours incubation at 37°C. with shaking for Klebsiella baumannii and Acinetobacter baumannii, at34° C. without shaking for Pseudomonas aeruginosa, the growth inhibitionwas measured by OD₆₀₀. Results are provided in Table 1.

TABLE 1 P. K. A. Sample aeruginosa pneumoniae baumannii mono (N^(γ)-bis-4 μg/ml 8 μg/ml 4 μg/ml sulfomethyl) Polymyxin E1 Colistin 2 μg/ml 4μg/ml 0.5 μg/ml   CMS 4 μg/ml 8 μg/ml 4 μg/ml

Example 6: Toxicity of the Compounds of the Present Invention

Nephrotoxicity was measured using KIM-1 (kidney injury molecule-1) as abiomarker of acute tubular necrosis.

Mice were treated with a mixture comprising 93%mono(N^(γ)-bis-sulfomethyl) Polymyxin E1 or CMS for 5 days and weremonitored for clinical signs of toxicity and urine concentrations ofKIM-1. During the 5 days of treatment mice showed no clinical signs oftoxicity. KIM-1 levels in urine indicated a slight increase compared tovehicle treatment. The normal range of KIM-1 in mouse urine, as statedin the ELISA kit instruction, is 456-8048 pg/ml.

The mixtures were received as dry powder and were dissolved in the vialsin sterile saline to the concentration stated in table 1, immediatelyprior to injection.

Once a day for 5 days, mice were treated subcutaneously in the neckregion with 0.2 ml corresponding to the doses stated in Table 1. Thedosing was based on a mean weight of 28 g.

Urine samples were collected at 24 hrs after the 2nd dosing and 4 hrsafter the 5th dosing. Samples were stored at −80° C. until analysed forKIM-1 concentrations

Urine samples were thawed, centrifuged for 5 min at 2000 g and diluted×50 prior to performing the ELISA according the instruction in theQuantikine ELISA mouse TIM-1/KIM-1/HAVCR Immunoassay kit.

TABLE 1 Treatment schedule Concentration of Treatment: Dose dosingsolution Number of Mice A mixture comprising 3.5 mg/ml 5 93% mono(N^(γ)-bis- sulfomethyl) Polymyxin E1: 25 mg/kg a mixture comprising 93%4.3 mg/ml 5 mono (N^(γ)-bis-sulfomethyl) Polymyxin E1: 30 mg/kg CMS: 32mg/kg 4.7 mg/ml 5 Gentamicin: 80 mg/kg  12 mg/ml 5 Vehicle 5

All the tested mixtures were well tolerated by mice as determined bybody weight and clinical scoring. Thickened skin at the site ofinjection was observed in the groups of mice injected with a mixturecomprising 93% mono (N^(γ)-bis-sulfomethyl) Polymyxin E1. However, onlya slight, but not significant, increase in urine KIM-1 levels wasobserved:

TABLE 2 Urinary KIM-1 concentrations after 5 doses. Treatment Mouse idKIM-1 (pg/ml) A mixture comprising 93% mono (N^(γ)- 31 5400bis-sulfomethyl) Polymyxin E1 32 4700 25 mg/kg 33 1600 34 9500 35 7350 Amixture comprising 93% mono (N^(γ)- 36 6800 bis-sulfomethyl) PolymyxinE1 37 3200 30 mg/kg 38 8900 40 5500 CMS32 mg/kg 41 12700 42 4350 43 195044 3800 45 3800 Gentamicin 80 mg/kg 46 12850 47 5900 48 5750 Vehicle 516950 52 5400 54 2500

Example 6: In Vivo Activity of Mono Bis-Sulfomethylated Polymyxin E1

A mouse model with pulmonary infections of Pseudomonas aeruginosa(strain PAX11045) were treated with peritoneal administration ofPolymyxin E1, CMS E1 and mono bis-sulfomethylated Polymyxin E1. CFU isthe calculated number of colony forming units in the lung tissue after22 hours except for the non-treatment group of mice. The clinicalscoring is based on observations at the respective time points, as shownin table 3:

TABLE 3 log10 mean Clinical Clinical CFU log10 score at score atTreatment lung CFU T = 19 T = 22 None 6.00 6.10 T = 4 6.08 6.06 6.196.15 Polymyxin- 5.65 4.43 3 4 E1 (dose: 4.08 2 2 10 mg/kg) 6.06 3 4 4.053 3 3.78 3 3 3.70 3 3 3.72 3 3 A mixture 6.67 5.60 4 comprising 7.17 4mono (N_(γ)- 6.13 4 bis- 5.14 3 3 sulfomethyl) 4.63 3 3 Polymyxin 3.83 33 E1 (dose: 51 mg/kg) CMS E1 3.95 4.53 2 2 (dose: 41 5.40 4 mg/kg) 3.983 3 4.60 3 3 4.65 3 3 4.60 3 3 Vehicle 7.05 6.80 3 5 7.15 3 5 7.13 3 56.35 3 4 6.57 3 4 6.05 3 4 7.30 3 4 Score 0 healthy Score 1 minorclinical signs of infection and inflammation like piloerection in theskin, distress and social withdrawal Score 2 moderate signs of infectionlike changed body position, changes in pattern of movement, lack ofcuriosity or changed activity. Score 3 severe signs of infection likestiff movements, forced ventilation, Score 4 severe pain, mouse wassacrificed immediately to minimize suffering Score 5 The mouse was dead

The invention claimed is:
 1. A polymyxin represented by formula (I):

wherein R¹ is an aliphatic linear or branched C₆-C₁₀ acyl group, or

R⁵ is —CH(CH₃)₂, —CH₂CH(CH₃)₂, —CH(CH₃)CH₂CH₃, or —CH₂C₆H₅; R⁶ is—CH(CH₃)₂, —CH₂CH(CH₃)₂, or —CH(CH₃)CH₂CH₃; each of R², R³, R⁴, R⁷ andR⁸ is either —(CH₂)_(x)CH₂NH₂ or —(CH₂)_(x)CH₂N(CH₂SO₃M)₂; wherein x is0 or 1; wherein M is a cation; and wherein one of R², R³, R⁴, R⁷ and R⁸is —(CH₂)_(x)CH₂N(CH₂SO₃M)₂, and the remaining four of R², R³, R⁴, R⁷and R⁸ are —(CH₂)_(x)CH₂NH₂.
 2. The polymyxin of claim 1, wherein R¹ isan aliphatic linear or branched C₆-C₁₀ acyl group.
 3. The polymyxin ofclaim 2, wherein R¹ is heptanoyl, 6-methylheptanoyl, octanoyl,(S)-6-methyloctanoyl, 3-hydroxy-6-methyloctanoyl, 7-methyloctanoyl,nonanoyl, 8-methylnonanoyl or decanoyl.
 4. The polymyxin of claim 1,wherein M is a pharmaceutically acceptable monovalent cation.
 5. Acomposition comprising more than 50% as measured by HPLC of thepolymyxin of claim
 1. 6. A composition comprising more than 80% asmeasured by HPLC of the polymyxin of claim
 1. 7. A compositioncomprising more than 90% as measured by HPLC of the polymyxin ofclaim
 1. 8. A method of treating an infection caused by a Gram-negativebacterium, comprising contacting the bacterium with the polymixin ofclaim 1.